The mechanisms that orchestrate and/or perpetuate chronic airway inflammation, believed to be key factors in the progression of asthma, remain unknown. Recent evidence suggests that airway smooth muscle (ASM) synthetic function, defined as secretion of cytokines, chemokines or growth factors and expression of adhesion molecules, may play an active role in regulating airway inflammation in asthma. In the previous funding period, we identified multiple transcription factors such as NF-kappaB and NF-AT that regulate cytokine-induced inflammatory gene expression in human ASM cells. In our new preliminary studies, we found that TNFalpha activates other transcription factors IRF-1, STAT1 and STAT2, and differentially regulates the expression of CD38, IL-6, RANTES and eotaxin but not ICAM-1 via the autocrine action of secreted IFNbeta. Accordingly, the aims of this COMPETING CONTINUATION proposal seek to extend our previous findings by defining the relative contribution of each of these signaling molecules in the regulation of TNFalpha effects on synthetic functions. The central hypothesis of this proposal states that TNFalpha and autocrine IFNbeta act in synergy to differentially regulate inflammatory gene expression via the coordinated activation of STAT1/STAT2, NF-kappaB and CD38-dependent pathways. In Aim 1, we will determine whether NF-kappaB functionally interacts with STAT1/STAT2 and IRF-1 to regulate TNFalpha-induced expression of inflammatory genes in an IFNbeta-dependent manner using cytokine promoter constructs and coimmunoprecipitation, co-localization and gel shift assays. In Aim 2, we will identify the transcriptional mechanisms (TRAFIKK) regulating cytokine-induced IFNbeta expression in human ASM cells using dominant negative and constitutively active cDNA constructs. We will also determine the in vivo relevance of IFNbeta expression by ASM in a murine model of bronchial hyper-responsiveness by characterizing the time course of in vivo expression and/or activation of IFNbeta signaling molecules in ASM. In Aim 3, we will characterize whether IFNbeta-dependent CD38 pathways are necessary and/or sufficient to regulate TNFalpha-induced expression of inflammatory genes in ASM cells. The role of CD38 will be investigated using CD38 -/- ASM and soluble inhibitors. The role of calcium-dependent pathways in TNFalpha-inducible genes will be assessed by examining the role of FKBP12.6 and NF-AT using gene-deficient mice, reporter constructs and soluble inhibitors. Because of the importance of inflammatory cytokines/chemokines in asthma, results from our studies will determine that ASM-derived cytokines participate in the regulation of airway inflammation not only via paracrine effects but also via the autocrine modulation of ASM synthetic function. Thus, understanding the transcriptional mechanisms regulating the expression of inflammatory genes in ASM will likely lead to new therapeutic approaches for the treatment of asthma.